Yielding behaviour of chemically treated Pseudomonas fluorescens biofilms

The mechanics of biofilms are intrinsically shaped by their physicochemical environment. By understanding the influence of the extracellular matrix composition, pH and elevated levels of cationic species on the biofilm rheology, novel living materials with tuned properties can be formulated. In this study, we examine the role of a chaotropic agent (urea), two divalent cations and distilled deionized water on the nonlinear viscoelasticity of a model biofilm Pseudomonas fluorescens. The structural breakdown of each biofilm is quantified using tools of non-linear rheology. Our findings reveal that urea induced a softening response, and displayed strain overshoots comparable to distilled deionized water, without altering the microstructural packing fraction and macroscale morphology. The absorption of divalent ferrous and calcium cations into the biofilm matrix resulted in stiffening and a reduction in normalized elastic energy dissipation, accompanied by macroscale morphological wrinkling and moderate increases in the packing fraction. Notably, ferrous ions induced a predominance of rate dependent yielding, whereas the calcium ions resulted in equal contribution from both rate and strain dependent yielding and structural breakdown of the biofilms. Together, these results indicate that strain rate increasingly becomes an important factor controlling biofilm fluidity with cation-induced biofilm stiffening. The finding can help inform effective biofilm removal protocols and in development of bio-inks for additive manufacturing of biofilm derived materials.

) can be calculated as tangent to the elastic LB plot at zero strain.Large strain modulus (G ′ L ) can be calculated as slope of the line from the origin to the point of maximum strain on a elastic LB curve.(b) A plot of stress vs strain rate at constant frequency, also known as viscous LB plot.Minimum strain viscosity (η ′ M ) can be calculated as tangent to the viscous LB plot at zero strain rate.Large strain modulus (η ′ L ) can be calculated as slope of the line from the origin to the point of maximum strain rate on a viscous LB curve.Figure S2: Figure shows an elastic Lissajous Bowditch (LB) plot that maps the intracyle stress and strain from during an oscillatory cycle.The residual modulus G R is calculated as the differential of stress with respects to strain at the point of zero stress.Alternatively, G R can be interpreted as tangent to the LB plot at the point where stress is zero.The accumulated strain (γ accumulated ) shown by light green curve, is calculated as the total stain accumulated between the lower reversal point at which strain becomes positive (the blue dot), to the point of maximum stress (the red dot).

Figure S1 :
Figure S1: Figure shows (a) A plot of stress vs strain at constant frequency, also known as elastic Lissajous Bowditch (LB) plot.Minimum strain modulus (G ′M ) can be calculated as tangent to the elastic LB plot at zero strain.Large strain modulus (G ′ L ) can be calculated as slope of the line from the origin to the point of maximum strain on a elastic LB curve.(b) A plot of stress vs strain rate at constant frequency, also known as viscous LB plot.Minimum strain viscosity (η ′ M ) can be calculated as tangent to the viscous LB plot at zero strain rate.Large strain modulus (η ′ L ) can be calculated as slope of the line from the origin to the point of maximum strain rate on a viscous LB curve.

Figure S5 :
Figure S5: Viscoelasticity of biofilms grown on an agar plate with no treatments added (a) Amplitude sweep.(b) Frequency sweep , n ≥ 3. Error bar indicates standard deviation of mean.

Figure S7 :
Figure S7: Viscous LB plots showing the presence of intersecting loops (red arrows) for chemically treated biofilms.

Figure S8 :
Figure S8: Normalised energy dissipated (E norm = E/E(γ = 0.13%)) as a function of applied strain.Dissipated energy (E) values were obtained by calculating area enclosed by the elastic LB plots for the applied strain (γ) and divided by the dissipated energy at γ = 0.133%.n ≥ 3. error bar indicates standard deviation of mean.

Table S1 :
pH of the chemical solutions applied to the biofilms.